SUMMARY Acquired resistance to targeted therapies in cancer is a rising unmet clinical need. In prostate cancer (PCa), the success of next-generation androgen receptor (AR) pathway inhibitors have been hampered by the development of drug resistance. This acquired resistance involves, in many cases, the reactivation of the AR axis through several different mechanisms. However, it has become apparent that an alternative mechanism of resistance is driven by the reprogramming of prostate cancer cells to undergo lineage plasticity to adopt an AR-independent state and to acquire a neuroendocrine phenotype, that allow them to grow and survive and escape AR-therapy. More potent and sustained AR targeting has driven an increased incidence of neuroendocrine prostate cancer (NEPC), which is an extremely aggressive, highly proliferative and metastatic PCa variant. Therefore, understanding the molecular mechanisms that govern NEPC differentiation is a pressing unmet clinical need. Our preliminary data have identified protein kinase C (PKC)l/i as a novel tumor suppressor in NEPC. Our recently published data demonstrate that the kinase PKCl/i is downregulated in human NEPC patients, and its loss promotes a metabolic reprogramming that sustains increased proliferation, as well as epigenetic changes needed by PCa cells to undergo cancer cell plasticity towards NEPC differentiation. The loss of PKCl/i results in the upregulation of the serine and one-carbon pathway metabolism that leads to increased production of S- adenosine methionine (SAM), which is the methyl donor for DNA and histone methylation. Our new unpublished data demonstrate that PKCl/i, in addition to be critical to produce SAM, also directly regulates DNMT1 and EZH2, key methyltransferases that utilize SAM, and are the ultimate chromatin modifiers. Therefore, our overarching hypothesis is that PKCl/i by regulating not only SAM generation but also the epigenetic modifiers of the PCa genome during NEPC differentiation creates new vulnerabilities that can be exploited therapeutically. Three key questions will be addressed in this proposal: (Aim 1) How does PKCl/i-mediated phosphorylation control EZH2 and DNMT1 functions during NEPC differentiation? (Aim 2) How does DNMT1 and EZH2- dependent epigenetic reprogramming downstream of PKCl/i contribute to the acquisition of the different NEPC traits? (Aim 3) Is PHGDH (the limiting enzyme in serine biosynthesis) inhibition alone or in combination with epigenetic inhibitors an effective therapeutic approach for treating NEPC tumors? The successful completion of this proposal will allow us to advance our understanding of the molecular mechanisms governing lineage plasticity during NEPC differentiation and help in the identification of new vulnerabilities that could lead to novel therapies in this lethal PCa.